Syndecan-4 is essential for development of concentric myocardial hypertrophy via stretch-induced activation of the calcineurin-NFAT pathway.

Sustained pressure overload leads to compensatory myocardial hypertrophy and subsequent heart failure, a leading cause of morbidity and mortality. Further unraveling of the cellular processes involved is essential for development of new treatment strategies. We have investigated the hypothesis that the transmembrane Z-disc proteoglycan syndecan-4, a co-receptor for integrins, connecting extracellular matrix proteins to the cytoskeleton, is an important signal transducer in cardiomyocytes during development of concentric myocardial hypertrophy following pressure overload. Echocardiographic, histochemical and cardiomyocyte size measurements showed that syndecan-4(-/-) mice did not develop concentric myocardial hypertrophy as found in wild-type mice, but rather left ventricular dilatation and dysfunction following pressure overload. Protein and gene expression analyses revealed diminished activation of the central, pro-hypertrophic calcineurin-nuclear factor of activated T-cell (NFAT) signaling pathway. Cardiomyocytes from syndecan-4(-/-)-NFAT-luciferase reporter mice subjected to cyclic mechanical stretch, a hypertrophic stimulus, showed minimal activation of NFAT (1.6-fold) compared to 5.8-fold increase in NFAT-luciferase control cardiomyocytes. Accordingly, overexpression of syndecan-4 or introducing a cell-permeable membrane-targeted syndecan-4 polypeptide (gain of function) activated NFATc4 in vitro. Pull-down experiments demonstrated a direct intracellular syndecan-4-calcineurin interaction. This interaction and activation of NFAT were increased by dephosphorylation of serine 179 (pS179) in syndecan-4. During pressure overload, phosphorylation of syndecan-4 was decreased, and association between syndecan-4, calcineurin and its co-activator calmodulin increased. Moreover, calcineurin dephosphorylated pS179, indicating that calcineurin regulates its own binding and activation. Finally, patients with hypertrophic myocardium due to aortic stenosis had increased syndecan-4 levels with decreased pS179 which was associated with increased NFAT activation. In conclusion, our data show that syndecan-4 is essential for compensatory hypertrophy in the pressure overloaded heart. Specifically, syndecan-4 regulates stretch-induced activation of the calcineurin-NFAT pathway in cardiomyocytes. Thus, our data suggest that manipulation of syndecan-4 may provide an option for therapeutic modulation of calcineurin-NFAT signaling.

Inhibition of pulmonary fibrosis in mice by CXCL10 requires glycosaminoglycan binding and syndecan-4.

Pulmonary fibrosis is a progressive, dysregulated response to injury culminating in compromised lung function due to excess extracellular matrix production. The heparan sulfate proteoglycan syndecan-4 is important in mediating fibroblast-matrix interactions, but its role in pulmonary fibrosis has not been explored. To investigate this issue, we used intratracheal instillation of bleomycin as a model of acute lung injury and fibrosis. We found that bleomycin treatment increased syndecan-4 expression. Moreover, we observed a marked decrease in neutrophil recruitment and an increase in both myofibroblast recruitment and interstitial fibrosis in bleomycin-treated syndecan-4-null (Sdc4-/-) mice. Subsequently, we identified a direct interaction between CXCL10, an antifibrotic chemokine, and syndecan-4 that inhibited primary lung fibroblast migration during fibrosis; mutation of the heparin-binding domain, but not the CXCR3 domain, of CXCL10 diminished this effect. Similarly, migration of fibroblasts from patients with pulmonary fibrosis was inhibited in the presence of CXCL10 protein defective in CXCR3 binding. Furthermore, administration of recombinant CXCL10 protein inhibited fibrosis in WT mice, but not in Sdc4-/- mice. Collectively, these data suggest that the direct interaction of syndecan-4 and CXCL10 in the lung interstitial compartment serves to inhibit fibroblast recruitment and subsequent fibrosis. Thus, administration of CXCL10 protein defective in CXCR3 binding may represent a novel therapy for pulmonary fibrosis.

Fibulin-5 accelerates elastic fibre assembly in human skin fibroblasts.

Fibulin-5 null mice display abnormalities in the elastic fibres in the dermis. We postulated, therefore, that fibulin-5 might be a regulator of elastic fibre assembly and stability. To clarify the role of fibulin-5 in elastic fibre formation, we employed in vitro systems that allowed increasing expression of elastic fibre components by gene transduction using retroviral vector constructs. First, the human tropoelastin gene (ELN) was transduced into human dermal fibroblasts, which resulted in elevated gene expression. These cells were then cultured in monolayer, but the overexpression of ELN in this system did not alter the assembly of elastic fibres. However, incubation of fibroblasts with TGF-beta1 resulted in elastic fibre accumulation, and the expression of fibulin-5 was enhanced by TGF-beta1. Thus, we overexpressed human fibulin-5 cDNA in dermal fibroblasts using a retroviral vector containing a cytomegalovirus (CMV) promoter. These cells deposited elastic fibres. These results suggest that fibulin-5 is a critical component in the control of elastic fibre assembly by dermal fibroblasts.

Foxn1 promotes keratinocyte differentiation by regulating the activity of protein kinase C.

The transcription factor Foxn1 (the product of the nude locus) promotes the terminal differentiation of epithelial cells in the epidermis and hair follicles. Activated early in terminal differentiation, Foxn1 can modulate the timing or order of trait acquisition, as it induces early features of epidermal differentiation while suppressing late features. Here, we identify protein kinase C (PKC) as a key target of Foxn1 in keratinocyte differentiation control. Foxn1 has broad negative effects on the PKC family, as the loss of Foxn1 function leads to higher levels of total, primed, and activated PKC. Phosphorylated PKC substrates (the mediators of PKC function) rise when Foxn1 is inactivated and fall when Foxn1 is overproduced, suggesting that Foxn1 antagonizes PKC's effects. When PKC inhibitors are applied to nude (Foxn1 null) keratinocytes, nude defects are normalized or suppressed, as the inhibitors prevent nude cells from underproducing early differentiation markers and overproducing late markers. Taken together, the results suggest that Foxn1 acts as a restraint or brake on PKC signaling and that without this brake PKC disrupts differentiation. The results further suggest that Foxn1 modulates stage-specific markers by modulating PKC activity, providing control over the timing of steps in the differentiation program.

Syndecan-4 dependent FGF stimulation of mouse vibrissae growth.

The development, maintenance and regeneration of epithelial appendages such as hairs or vibrissae depend on reciprocal interactions between the epidermal and the dermal components of the integument. Growth factors are among a number of signaling molecules that have been identified during these developmental events. Growth factors such as fibroblast growth factors (FGFs) bind cell surface heparan sulfate proteoglycans (HSPGs) on their heparan sulfate side chains and as such these proteoglycans act as co-receptors for FGF receptors (FGFRs) by forming a ternary signaling complex of HSPG, FGFR and FGF. The syndecans make up a family (syndecan-1-4) of transmembrane HSPGs. In the present study we examined the growth response of mouse vibrissae to HSPG-binding growth factors as a function of the presence or absence of syndecan-4 in an organ culture system. Syndecan-4 is expressed on keratinocytes that make up the inner root sheath of the vibrissa. Vibrissae from wild-type mice, but not from syndecan-4 null mice, displayed a statistically significant and dose-dependent growth response to FGF-1, FGF-2 and FGF-7. In contrast, a statistically significant growth response is seen in vibrissae from both wild-type and syndecan-4 null mice when the culture medium is supplemented with either hepatocyte growth factor (HGF) that binds to HSPG, insulin that does not bind to HSPG or 5% fetal bovine serum. The syndecan-4 dependent effect of FGF-1, -2 and -7 on the transcriptional activity of IRS expressed genes and of genes involved in cell proliferation reveals a number of different response patterns. In vivo, the vibrissae of syndecan-4 null mice are shorter and have a smaller diameter than those of wild-type mice and this phenotype may result from a suboptimal response to growth factors. Syndecan-1, which is expressed in the outer root sheath of the vibrissae shaft, does not influence the response of the vibrissae to FGF-1, -2 and -7 and the length and diameter of vibrissae of syndecan-1 null mice do not differ from those of wild-type mice.

Matrix contraction by dermal fibroblasts requires transforming growth factor-beta/activin-linked kinase 5, heparan sulfate-containing proteoglycans, and MEK/ERK: insights into pathological scarring in chronic fibrotic disease.

Scarring is characterized by excessive synthesis and contraction of extracellular matrix. Here, we show that fibroblasts from scarred (lesional) areas of patients with the chronic fibrotic disorder diffuse scleroderma [diffuse systemic sclerosis (dSSc)] show an enhanced ability to adhere to and contract extracellular matrix, relative to fibroblasts from unscarred (nonlesional) areas of dSSc patients and dermal fibroblasts from normal, healthy individuals. The contractile abilities of normal and dSSc dermal fibroblasts were suppressed by blocking heparin sulfate-containing proteoglycan biosynthesis or antagonizing transforming growth factor-beta receptor type I [activin-linked kinase (ALK5)] or ras/mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK). Compared with both normal and nonlesional fibroblasts, lesional dSSc fibroblasts overexpressed the heparin sulfate-containing proteoglycan syndecan 4. We also found that the procontractile signals from transforming growth factor (TGF)-beta were integrated through syndecan 4 and MEK/ERK because the ability of TGFbeta to induce contraction of dermal fibroblasts was prevented by MEK antagonism. TGFbeta could not induce a contractile phenotype or phosphorylate ERK in syndecan 4(-/-) dermal fibroblasts. These results suggest that integrating TGFbeta and ERK signals via syndecan 4 is essential for the contractile ability of dermal fibroblasts. We conclude that antagonizing MEK/ERK, TGFbeta1/ALK5, or syndecan 4 may alleviate scarring in chronic fibrotic disease.

Phosphoinositide 3-kinase signaling to Akt promotes keratinocyte differentiation versus death.

Signaling pathways regulating the differentiation program of epidermal cells overlap widely with those activated during apoptosis. How differentiating cells remain protected from premature death, however, is still poorly defined. We show here that the phosphoinositide 3-kinase (PI3K)/Akt pathway is activated at early stages of mouse keratinocyte differentiation both in culture and in the intact epidermis in vivo. Expression of active Akt in keratinocytes promotes growth arrest and differentiation, whereas pharmacological blockade of PI3K inhibits the expression of "late" differentiation markers and leads to death of cells that would otherwise differentiate. Mechanistically, the activation of the PI3K/Akt pathway in keratinocyte differentiation depends on the activity of the epidermal growth factor receptor and Src families of tyrosine kinases and the engagement of E-cadherin-mediated adhesion. During this process, PI3K associates increasingly with cadherin-catenin protein complexes bearing tyrosine phosphorylated YXXM motifs. Thus, the PI3K signaling pathway regulates the choice between epidermal cell differentiation and death at the cross-talk between tyrosine kinases and cadherin-associated catenins.

FGF-2 signaling is sufficient to induce dermal condensations during feather development.

In a previous report, we showed that fibroblast growth factor-2 (FGF-2) is a signal produced by epidermal placode cells during feather development and that this growth factor can induce feathers in scaleless mutant skins that fail to form feathers due to a defective epidermis (Song et al., [1996] Proc Natl Acad Sci USA 93:10246-10249). Here, we test whether FGF-2 is sufficient to induce dermal condensations, structures that normally form under the control of signals from the epidermal placode and are identified on the basis of aggregation of cells, the expression of FGF receptor-1 and bone morphogenetic protein-2 transcripts and the cessation of proliferation of the condensed cells. By using denuded 8-day scaleless dermis as a test system, we have established that FGF-2 is sufficient to induce dermal condensation. We suggest that the primary effect of FGF-2 is an increase in cellular density mediated through cell migration, followed by the expression of dermal condensation-specific genes and cessation of cell proliferation. The FGF-2 effect can be abolished by heparin, suggesting the involvement of heparan sulfate proteoglycans (HSPGs) in growth factor signaling. The spatiotemporal expression of syndecan-3 during feather development suggests that this cell-surface HSPG may be involved in the response of competent embryonic skin dermis to FGF-2.

Syndecan-4 regulates ATF-2 transcriptional activity in a Rac1-dependent manner.

Syndecan-4 is a transmembrane heparan sulfate proteoglycan that co-operates with integrins during cell-matrix interactions for the assembly of focal adhesions and actin stress fibers and in the phosphorylation of focal adhesion kinase (FAK) on Tyr397. These cellular events are regulated by the small GTPase Rho, and in the absence of syndecan-4 ligation, cellular levels of GTP-bound Rho are decreased implicating syndecan-4 in the regulation of the small GTPases. In the present study we report that, compared with wild type cells, fibronectin-adherent syndecan-4-null fibroblasts showed enhanced lamellipodia and increased Rac1 activity that could be down-regulated by re-expression of syndecan-4 in the mutant cells. Consistent with the role for Rac1 in activating p38 and JNK signaling, syndecan-4-null cells display higher levels of active p38 MAPK and JNK that were abolished by the expression of a dominant-negative RacN17 mutant. Since p38 and JNK regulate gene expression by phosphorylating and activating transcription factors, we compared both the phosphorylation state and the transcriptional activity of the ATF-2 transcription factor, as a direct p38 and JNK target in syndecan-4-null and wild type cells. In the absence of syndecan-4, both ATF-2 phosphorylation and transcriptional activity were significantly more elevated compared with wild type cells, and both activities were decreased either by the re-expression of syndecan-4 or by the expression of RacN17. Our results reveal a novel function for syndecan-4 in modulating nuclear transcriptional activity and indicate an underlying mechanism that acts at the level of Rac1-p38/JNK signaling.

Essential and separable roles for Syndecan-3 and Syndecan-4 in skeletal muscle development and regeneration.

Syndecan-3 and syndecan-4 function as coreceptors for tyrosine kinases and in cell adhesion. Syndecan-3(-/-) mice exhibit a novel form of muscular dystrophy characterized by impaired locomotion, fibrosis, and hyperplasia of myonuclei and satellite cells. Explanted syndecan-3(-/-) satellite cells mislocalize MyoD, differentiate aberrantly, and exhibit a general increase in overall tyrosine phosphorylation. Following induced regeneration, the hyperplastic phenotype is recapitulated. While there are fewer apparent defects in syndecan-4(-/-) muscle, explanted satellite cells are deficient in activation, proliferation, MyoD expression, myotube fusion, and differentiation. Further, syndecan-4(-/-) satellite cells fail to reconstitute damaged muscle, suggesting a unique requirement for syndecan-4 in satellite cell function.

Syndecan-4 modulates focal adhesion kinase phosphorylation.

The cell-surface heparan sulfate proteoglycan syndecan-4 acts in conjunction with the alpha(5)beta(1) integrin to promote the formation of actin stress fibers and focal adhesions in fibronectin (FN)-adherent cells. Fibroblasts seeded onto the cell-binding domain (CBD) fragment of FN attach but do not fully spread or form focal adhesions. Activation of Rho, with lysophosphatidic acid (LPA), or protein kinase C, using the phorbol ester phorbol 12-myristate 13-acetate, or clustering of syndecan-4 with antibodies directed against its extracellular domain will stimulate formation of focal adhesions and stress fibers in CBD-adherent fibroblasts. The distinct morphological differences between the cells adherent to the CBD and to full-length FN suggest that syndecan-4 may influence the organization of the focal adhesion or the activation state of the proteins that comprise it. FN-null fibroblasts (which express syndecan-4) exhibit reduced phosphorylation of focal adhesion kinase (FAK) tyrosine 397 (Tyr(397)) when adherent to CBD compared with FN-adherent cells. Treating the CBD-adherent fibroblasts with LPA, to activate Rho, or the tyrosine phosphatase inhibitor sodium vanadate increased the level of phosphorylation of Tyr(397) to match that of cells plated on FN. Treatment of the fibroblasts with PMA did not elicit such an effect. To confirm that this regulatory pathway includes syndecan-4 specifically, we examined fibroblasts derived from syndecan-4-null mice. The phosphorylation levels of FAK Tyr(397) were lower in FN-adherent syndecan-4-null fibroblasts compared with syndecan-4-wild type and these levels were rescued by the addition of LPA or re-expression of syndecan-4. These data indicate that syndecan-4 ligation regulates the phosphorylation of FAK Tyr(397) and that this mechanism is dependent on Rho but not protein kinase C activation. In addition, the data suggest that this pathway includes the negative regulation of a protein-tyrosine phosphatase. Our results implicate syndecan-4 activation in a direct role in focal adhesion regulation.

Overexpression of serpin squamous cell carcinoma antigens in psoriatic skin.

Squamous cell carcinoma antigen belongs to the serpin family and is used for the diagnosis and management of squamous cell carcinoma. We investigated the involvement of squamous cell carcinoma antigen in psoriasis, as it is always detected in the sera of patients with psoriasis. Squamous cell carcinoma antigen localization in psoriatic epidermis varied depending on its concentration in the patient's sera. When its level was low in serum, weak and scattered staining was observed in the granular layer. With a high concentration of squamous cell carcinoma antigen, strong staining through the suprabasal to granular layer and condensed staining around the plasma membrane or intracellular space was detected in the affected epidermis. Interestingly, squamous cell carcinoma antigen was abundant in nuclei of the granular layer cells and elongated rete ridges. Immunoelectron microscopy confirmed the localization of squamous cell carcinoma antigen in the nuclei as well as in the periphery of the cell membrane. A cDNA library was constructed from psoriatic epidermis and both clones, SCCA1 and SCCA2, were obtained. Attempts to raise specific antibodies or to prepare cRNA probes for SCCA1 and SCCA2 were unsuccessful because of their nearly identical structures. A primer pair from each reactive site sequence enabled us to give a distinctive product for SCCA1 and SCCA2 by reverse transcription polymerase chain reaction. Analysis using these primers demonstrated that the SCCA2 transcript was specifically expressed in psoriatic skin tissues. Our results suggest that overexpression of squamous cell carcinoma antigens is associated with the disease activity of psoriasis.

Syndesmos, a syndecan-4 cytoplasmic domain interactor, binds to the focal adhesion adaptor proteins paxillin and Hic-5.

Syndecan-4 and integrins are the primary transmembrane receptors of focal adhesions in cells adherent to extracellular matrix molecules. Syndesmos is a cytoplasmic protein that interacts specifically with the cytoplasmic domain of syndecan-4, and it co-localizes with syndecan-4 in focal contacts. In the present study we sought possible interactors with syndesmos. We find that syndesmos interacts with the focal adhesion adaptor protein paxillin. The binding of syndesmos to paxillin is direct, and these interactions are triggered by the activation of protein kinase C. Syndesmos also binds the paxillin homolog, Hic-5. The connection of syndecan-4 with paxillin through syndesmos parallels the connection between paxillin and integrins and may thus reflect the cooperative signaling of these two receptors in the assembly of focal adhesions and actin stress fibers.

Syndecan-4: dispensable or indispensable?

Studies examining the role of the cell-surface heparan sulfate proteoglycan syndecan-4 have yielded a plethora of information regarding its role in both cell-matrix and growth-factor mediated signaling events. Many of the initial conclusions drawn from such research placed syndecan-4 in a keystone position within various signaling pathways though the generation of syndecan-4 null mice have surprised many in the field by indicating otherwise. These contradictory results place researchers in the frustrating and yet exhilarating position of still asking the question, "What role does syndecan-4 play in life?"